JPS6317840B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to novel phosphorylated nonapeptides with valuable biological and pharmacological properties and methods for their production. The new compound has the general formula This is a phosphorylated nonapeptide represented by Sequence L-Trp-L-Ala-Gly-Gly-L-Asp
-L-Ala-L-Ser-Gly-L-Glu (MW=
849), hereinafter referred to as DSIP (delta sleep-inducing peptide), was isolated from rabbit blood as a humoral sleep factor between 1971 and 1977, purified and characterized. The decision was made. Its structure was also finally elucidated (Pflu¨gers Arch. 369 ,
99−101, 1977). Biological studies conducted using synthetic DSIP have shown that synthetic DSIP, when administered intracerebral ventricularly to domestic rabbits, has the same effect as the natural compound isolated from rabbit blood.
gave the same results compared to DSIP (Experientia
33 , 548, 1977). An intracerebroventricular dose of 6 nMol DSIP/Kg induced biological effects in domestic rabbits that appeared in the EEG (electroencephalogram) to be equivalent to the phenomena found during natural orthodox deep sleep periods ( Proc.Natl.Acad.Sci.USA74, 1282
−1286, 1977). Synthetically produced DSIP is also active in cats and rats when administered intravenously at 30 nMol/Kg, allowing EEG recordings and passive behavioral tests to demonstrate significant effects in deep and paradoxal sleep. showed improvement. Synthesis by arterial perfusion of isolated rat heads
In animal studies, administration of DSIP also gave EEG recordings comparable to sleep, and the method of application, which can also be used in human medicine, induced a natural sleep state. However, in a series of experiments, the induction of sleep following administration of DSIP showed that the dose was slightly
It has been found that when changing by 20 nMol/Kg body weight, either a sharp effect or no effect occurs at all, ie only a precise dose of the correct amount of DSIP will achieve the desired effect. Furthermore, intravenous administration requires a dose of 6 nMol/Kg, which is substantially higher than that required for intracerebroventricular administration to induce sleep, and furthermore, when administration is performed by the intravenous route. It was found that the induction of sleep was delayed. These differences in activity according to the two types of applications mentioned above are not surprising. This is because it is known from experience that DSIP-type oligopeptides are rapidly degraded by enzymes when applied intravenously (subcutaneously or transdermally) and do not easily penetrate the blood-brain barrier. Because there is. It was therefore an object of the present invention to find compounds of the DSIP type that are more stable against enzymatic degradation, more easily penetrate the blood-brain barrier and have improved biological and pharmacological properties. be. According to a comparative study, DSIP, in which the OH group of serine is phosphorylated, has the following effects when administered intravenously to rats:
It has been shown to not only result in a reduction in the required dosage compared to DSIP, but also to provide enhanced anti-inflammatory activity with a more rapid onset and longer, i.e. longer duration. ing. This sustained and enhanced activity constitutes an important therapeutic advantage. Compounds according to the invention have the formula L-Trp-L-Ala-Gly-Gly-L-Asp-L-
The nonapeptide of Ala-L-Ser-Gly-L-Glu () is phosphorylated or the formula () in which at least one functional group is protected
It can be produced by a method characterized by removing a protecting group from a compound. The individual reactions leading to the final products of the invention and their purification as well as the preparation of the starting materials can be carried out using conventional methods, i.e. using methods well known in peptide chemistry. [For example, “Methoden der organischen
Chemie” (Houben-Weyl) Vol.XV parts 1 and 2, Georg Thieme Verlag, Stuttgart, 1974,
reference]. Thus, for example, synthetically produced
DSIP or OH of serine to be phosphorylated
One of the compounds of formula () in which a functional group other than the group is protected can be used as a starting material. Phosphorylation can be carried out by reaction with monofunctional derivatives of phosphoric acid such as dibenzylphosphoryl chloride or diphenylphosphoryl chloride. The protecting groups should be chosen so that they can be removed under mild conditions that do not result in changes to the peptide sequence. Examples of protecting groups that can be used include N
-benzyloxycarbonyl, N-benzyl-,
Included are benzyl ester and ether groups. This is because they can be easily removed by catalytic hydrogenolysis. Protected serine peptides can be phosphorylated using dibenzylphosphoryl chloride in anhydrous pyridine. The resulting phosphate derivative is washed, purified with aqueous acid or base and subjected to hydrogenolysis in t-butanol solution. The resulting phosphorylated peptides can be purified by thin layer or anion exchange chromatography. Phosphorylation of serine-containing peptides with diphenylphosphoryl chloride is preferred in that N-benzyloxycarbonyl- and benzyl ester groups are selectively eliminated by hydrogenolysis using a palladium catalyst. A platinum catalyst can be used to remove one or both phenyl groups [Acta.
Chem.Scan. 13 , 1407 and 1422 (1959)]. On the other hand, one starts with an unprotected DSIP sequence and converts this into a monohalogenophosphoric acid [e.g.
It can also be phosphorylated with CIPO(OH) ₂ . Next, the method for producing the phosphorylated nonapeptide of the present invention will be explained in more detail with reference to Examples. Example 1 Phosphorylation of N-benzyloxycarbonyl-DSIP-benzyl ester N-benzyloxycarbonyl-DSIP- dissolved in 10 ml of pyridine dried over barium oxide
A solution of benzyl ester (5 nMol) was cooled until almost frozen. Dibenzylphosphoryl chloride, freshly prepared from dibenzyl, was then added and the mixture was shaken well and left overnight at 4°C. Then 75 ml of cold ethyl acetate and 75 ml of cold water were added to the mixture and centrifuged. The supernatant water was subsequently washed with cold water, 1M H ₂ SO ₄ , H ₂ O, saturated NaHCO ₃ solution and water, and dried over anhydrous Na ₂ SO ₄ .
The phosphate ester of the protected peptide was obtained in solid form after vacuum distillation of the solvent. The solid was then dissolved in a t-butanol/water mixture and hydrogenated using 10% Pd/C.
The reaction mixture was filtered, the catalyst and liquid were washed with water, and the combined washings were evaporated to dryness under vacuum.
DSIP in which the hydroxy group of serine obtained in this way is phosphorylated

[Formula] was purified using thin layer chromatography or an anion exchanger [Acta.Chem.Scan. 15 , 163 (1961). ] Example 2 Phosphorylation of DSIP 5 mg of DSIP and 1 mg of L-tryptophan were mixed with 1 ml of phosphorus oxychloride. Add the mixture to concentrated formic acid.
The solution was dissolved in 100 ml, water was removed, and the mixture was stirred at 0°C for 8 hours. The product was then lyophilized over NaOH under high vacuum. After adding 2 ml of ice water, 1N
The pH of the solution was adjusted to 8 at 0°C using NaOH. This pH was held constant for 2 hours. Freeze-dry the solution again and add 0.5ml of the resulting freeze-dried product to water.
dissolved in it. A 100 μ aliquot of this solution was applied to a silica gel plate (layer thickness 2 mm, without binder) and developed with acetone/water (7:3 V/V) for 8 hours at room temperature. Cover the plate with aluminum foil except for the peripheral strip and add florescamine solution (0.2% in acetone)
was sprayed. Using ultraviolet light it was possible to determine the position of the band containing the desired substance (Rf0.47). These were scraped off and eluted with water.
The supernatant resulting from centrifugation of the eluate at 25000 g was lyophilized. The substance thus obtained
Dissolved in 0.5 ml H ₂ O and loaded onto a Sephadex-G15 column, then eluted with water. 1 ml fractions were collected. Fractions that showed UV absorption at 280 nm were collected and lyophilized. Substances purified and lyophilized in this way The rising test in rats is described below.
[J.Biol.Chem. 202 , 67
(1953)]. Example 3 20 mg DSIP in 3 ml phosphorus oxychloride
(23.5 μMol) was dissolved in 500μ formic acid at 0°C.
The reaction mixture is then mixed with complete exclusion of water.
Stirred at 0°C for 200 hours. Excess phosphorous oxychloride and formic acid were then removed under high vacuum. To remove the chlorine atom of the phosphate ester group, the above material was dissolved in 5 ml of ice water and incubated at 0-4°C for 2
The mixture was stirred at pH 8 for an hour. This material was finally lyophilized. The lyophilized product was then dissolved in 1.2 ml of water and applied to six plates for preparative thin layer chromatography (silica gel) in each case up to 2 cm from the edge of the plate. Development was carried out using acetone/water (7:3, V/V) for 8 hours at room temperature. The plates were covered with aluminum foil except for a 3 cm wide strip around the edges and sprayed with Florescamine solution (0.2% in acetone). Irradiation of the plate with UV light at 350 nm showed spots around the edges. The band with Rf value of 0.6 was marked, removed and eluted with water.
The eluate was centrifuged for 10 minutes at 20,000 g and the supernatant was lyophilized. The frozen peptide was dissolved in 2 ml of water, loaded onto a Sephadex G-15 column (220 ml), and eluted with water. The fractions were evaluated using 280 nm UV light and showed a peak with a flat shoulder. The fractions belonging to this peak were divided into three pools and lyophilized separately. Material from pools 1 and 2 was chromatographed on Sephadex G15 and lyophilized again. Pool 1 material was confirmed to be essentially 100% DSIP with the OH group of serine phosphorylated. It has an Rf value of 0.42 on silica gel plates in water/acetone (7:3 V/V) system. The non-phosphorylated starting material showed an Rf value of 0.87 under similar conditions. Yield 25%. Determination of Biological Activity in the Wakefulness Test Wakefulness was determined simultaneously in 8 test rats and 8 control animals in a randomized double-blind manner. In the awakening test, each awakening point of the test animal was counted. In the experiment, 0.2 ml of
Substance of the invention in NaCl solution (Test T) or NaCl
This was done for 90 minutes after intravenous injection of 0.2 ml of solution alone (Control C). Counting was again carried out by three unbiased, unbiased persons under randomized, double-blind conditions. The frequency of awakening of control animals was set as 100%, and the reduction in the number of awakenings of test animals was calculated. The results are summarized in the table below.

[Table] * Phosphorylation of serine group In addition, the acute toxicity (LD ₅₀ ) of the compound of the present invention in mice is 2 to 4 g/Kg iv. In a 6-week toxicity study, doses up to 6 mg/Kg/day did not produce any negative results in rats and dogs. The effective dosage range for humans of the compounds of this invention is generally 2-10 μg/Kg body weight (iv), preferably about 5 μg/Kg. Infusion should be done slowly and may be repeated up to 7 times a day. The compounds of the invention can be used in direct or delayed release pharmaceutical preparations of the active ingredients of the invention.
For example, water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc,
It can be used in admixture with organic or inorganic inert carrier substances suitable for enteral or parenteral administration, such as vegetable oils, polyalkylene glycols or yellow petrolatum. The pharmaceutical preparations can be in solid form, such as tablets, dragees or capsules, or in liquid form, such as solutions, suspensions or emulsions. If desired, they can be sterilized and/or contain auxiliary substances such as preservatives, stabilizers, wetting agents or emulsifiers, taste improvers, salts or buffers to alter the permeation pressure. The pharmaceutical composition of the present invention can be manufactured by conventional methods.

Claims (1)

[Claims] 1 formula A phosphorylated nonapeptide represented by 2 Formula L-Trp-L-Ala-Gly-Gly-L-Asp-L-
A formula characterized by phosphorylating a nonapeptide represented by Ala-L-Ser-Gly-L-Glu A method for producing a phosphorylated nonapeptide shown in 3. The method according to claim 2, wherein the phosphorylation is carried out by reaction with phosphoric acid, phosphoric acid ester, phosphoric acid halide, phosphoric acid halide ester or pyrophosphate. 4. The method according to claim 3, wherein the phosphorylation is carried out using chlorophosphoric acid. 5 formula A sleep-inducing agent characterized by containing a phosphorylated nonapeptide represented by the following as an active ingredient.